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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 318-326 |
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doi:10.1111/j.1745-7270.2006.00166.x |
Nuclear Factor-kB Signaling Pathway Constitutively
Activated in Esophageal Squamous Cell Carcinoma Cell Lines and Inhibition of Growth
of Cells by Small Interfering RNA
Fang TIAN, Wei-Dong ZANG,
Wei-Hong HOU, Hong-Tao LIU, and Le-Xun XUE*
Laboratory for Cell Biology,
Medical College, Zhengzhou University, Zhengzhou 450052, China
Received: January 11, 2006
Accepted: February 27, 2006
This study was supported by a grant from the �211 Project�
(Education Ministry of China 2002-2)
*Corresponding
author: Tel, 86-371-66658332; Fax, 86-371-66997182; E-mail,
[email protected]
Abstract������� Although constitutive nuclear
factor (NF)-kB activation has been reported
in many human tumors, the role of the NF-kB
pathway in esophageal squamous cell carcinoma (ESCC) has not been known. In
this study, NF-kB pathway in two ESCC cell
lines was investigated using immunocytochemistry, Western blot and reverse
transcription-polymerase chain reaction. The activation of NF-kB DNA binding was determined by electrophoretic
mobility-shift assay. RNA interference was used to specifically inhibit the
expression of p65. Growth of cells was evaluated by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results
showed that p50, p65, IkBa,
p-IkBa
and IkB kinase b were expressed and mainly localized in
the cytoplasm. Reverse transcription-polymerase chain reaction results showed
the constitutive expressions of p50, p65 and IkBa mRNA in the two ESCC cell lines.
Furthermore, the nuclear extracts revealed that p50 and p65 translocated to the
nucleus had DNA-binding activity. Finally, small interfering RNA of p65
decreased the expression of p65, and the viability of cells transfected with
p65 small interfering RNA was significantly suppressed at the same
concentration of 5-fluorouracil (P<0.05)
compared to untransfected cells. The results of this study showed that there
was the constitutively activated NF-kB
signaling pathway in the ESCC cell lines. RNA interference targeting at p65
increased the sensitivity of the ESCC cell lines to 5-fluorouracil, suggesting
that NF-kB might be a good target for
cancer treatment.
Key words������� NF-kB; RNA interference;
esophageal squamous cell carcinoma; IkBa; IkBa kinase
Esophageal cancer is one of the most frequently diagnosed carcinomas, ranked as the sixth most common cause of death among all cancers in the world [1], and is especially prevalent in China. Esophageal squamous cell carcinoma (ESCC) is still one of the most aggressive squamous cell cancers with poor prognosis and rapid progression [2]. Although therapy strategies have been improved, the prognosis of patients with ESCC is still not positive. The 5-fluorouracil (5-FU) is frequently used in combination therapy of esophageal cancer, but some patients have a poor response to 5-FU-based chemotherapy. Obviously, a better understanding of the molecular mechanisms of ESCC helps to refine therapy. Recently, much evidence has demonstrated that nuclear factor-kB (NF-kB/Rel) plays a critical role in carcinogenesis. NF-kB is a transcription factor discovered by Sen and Baltimore as a regulator of the expression of the k light chain of immunoglobulins in B cells [3]. The NF-kB/Rel family is composed of five subunits, p50/p105 (NF-kB1), p65 (RelA), C-Rel, P52/p100 (NF-kB2) and RelB, which can form various homo- or heterodimers. The most studied form is a heterodimer of the p50 and p65 subunits predominant in many types of cells [4].
In most normal cells, NF-kB is inactive by its tight association with the cytoplasmic inhibition proteins, named inhibitors of NF-kB (IkB), belonging to a gene family comprising IkBa, IkBb, IkBe, IkBg, Bcl-3, p100 and p105 [5]. A variety of extracellular stimulus factors, such as inflammatory cytokines, growth factors, DNA damaging agents, and bacterial and viral products, trigger a common signal transduction pathway based on the phosphorylation, ubiquitination and proteasome-dependent degradation of IkB to freely active NF-kB [6]. The released and activated NF-kB is rapidly translocated to the nucleus and binds to the promoter region in the relevant downstream genes to activate a series of transcriptional events. Thus, the phosphorylation of IkBa is an indispensable step to activate the NF-kB signaling pathway, which is catalyzed by an IkB kinase (IKK), a complex composed of IKKa, IKKb and IKKg. IKKb is the main catalytic subunit of the IKK complex in the phosphorylation of IkBa at two conserved serines (32 and 36) within the IkB N-terminal regulatory domain [7].
NF-kB has a key function in the transformation, proliferation and invasion of cancer cells as well as in resistance to radiotherapy and chemotherapy. Constitutively activated NF-kB has been detected in many human cancers including hepatocellular, colonic, pancreatic and cervical cancers [8-11]. However, the high activation status of NF-kB and its signaling pathway in ESCC has not been investigated. It has not been well understood whether or not this pathway is responsible for the proliferation in ESCC.
In this study, we investigated the mRNA and protein expression levels of certain members of the NF-kB signaling pathway, as well as NF-kB activity and the status of phosphorylation of IkBa in the two ESCC cell lines. The sensitivity to 5-FU in ESCC cells transfected with or without small interfering RNA (siRNA) targeting for p65 was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The results demonstrated that NF-kB was constitutively activated in the ESCC cell lines with the phosphorylation of the IkBa protein and overexpression of the genes. RNA interference (RNAi) targeting at p65 had anti-proliferative effects and increased the sensitivity of the ESCC cells to 5-FU.
Materials and Methods
Cell culture
Two human ESCC cell lines, Eca109 and EC9706, were provided by the State Key Laboratory of Molecular Oncology, Chinese Academy of Medical Science (Beijing, China), and cervical cancer cell line HeLa229 as the positive control cell was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Each of the three cell lines was cultured in RPMI 1640 medium (Gibco-BRL, Rockville, USA) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, USA), 100 U/ml penicillin and 100 mg/ml streptomycin at 37 �C with 5% CO2.
Antibodies and reagents
Mouse monoclonal antibodies to p65 (sc-8008), p-IkBa (sc-8404) and IKKb (sc-8014), and rabbit polyclonal antibodies to IkBa (sc-371) and p50 (sc-114) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Biotin 3' end labeling kit (89818) and LightShift chemiluminescent electrophoretic mobility shift assay (EMSA) kit (20148) were purchased from Pierce (Rockford, USA). Avian myeloblastosis virus (AMV) first strand DNA synthesis kit (BS252), Polymerase chain reaction (PCR) amplification kit (SK2491), oligonucleotides and primers were obtained from Shanghai Sangon Biological Engineering & Technology and Service (Shanghai, China). SignalSilence NF-kB p65 siRNA (6261) was purchased from Cell Signaling Technology (Beverly, USA).
Immunocytochemical analysis
The immunoreactivity was determined using the SP kit according to the manufacturer's protocols. Briefly, the three cell lines were plated on several glass slides and incubated at 37 �C with 5% CO2 for 24 h. The slides were rinsed three times in phosphate-buffered saline (PBS, pH 7.4), and fixed with 4% formaldehyde at room temperature (RT) for 10 min. After rinsing in PBS and treatment with 3% H2O2 for 10 min, the cells were blocked with 5% normal goat serum for 30 min in a humidified box at RT to eliminate non-specific binding, then incubated with antihuman p50, p65, IkBa and IKKb antibodies (1:100), p-IkBa (1:50), as well as PBS as the negative control, at 4 �C overnight. After the slides were rinsed three times in PBS and incubated with corresponding secondary antibodies for 30 min, they were developed with diaminobenzidine under a light microscope. Photomicrographs were taken immediately (magnification, 400).
Preparation of cytoplasm and
nuclear proteins
Cytoplasm and nuclear proteins were extracted from the cells cultured to approximately 90% confluence according to the instructions of the nuclear and cytoplasmic extraction reagents kit (Pierce). Aliquots of the proteins were stored at -70 �C and the protein concentrations were determined by Bradford method.
Western blot analysis
Cytoplasm (50 mg) from each cell line was added to 2�protein sample buffer, heated at 100 �C for 5 min, and separated using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), along with 20 ml of prestained protein molecular weight marker used as standard. Proteins were electrotransferred to supported nitrocellulose membranes (Amersham, Uppsala, Sweden) in transfer buffer containing 25 mM Tris, 193 mM glycine, and 20% methanol. The membranes were blocked in 5% skimmed milk in TBS-T (1�TBS, 0.05% Tween 20) at RT for 2 h, then rinsed three times in TBS-T and incubated with anti-p50, anti-p65, anti-IkBa, anti-P-IkBa and anti-IKKb antibodies, diluted in 1% skimmed milk, at RT for 2 h. The membranes were rinsed three times in TBS-T and incubated with 1:5000 dilution of goat antirabbit or goat antimouse secondary antibody conjugated to horseradish peroxidase for 1 h at RT. After extensive washing with TBS-T, proteins bands on the membranes were visualized by diaminobenzidine according to the manufacturer's instructions. Extracts (50 mg) from the nuclei were only detected in p50 and p65 proteins, as mentioned above.
EMSA
To determine NF-kB activation, NF-kB oligonucleotide using biotin 3'-end labeling was carried out by EMSA. Briefly, the NF-kB oligonucleotide 5'-AGTTGAGGGGACTTTCCCAGGC-3' DNA-binding sequence was labeled by the biotin 3' end labeling kit, and the nuclear proteins (10 mg) were incubated for 20 min at RT with biotin-labeled DNA probes in the 20 ml of reaction mixture comprising 25 mM EDTA, 5 mM MgCl2, 0.05% NP-40, 2.5% glycerol, 50 mg/ml poly(dI∙dC) and 0.2 mg bovine serum albumin. Nucleo-protein complexes were loaded onto the pre-electrophoresis 6% non-denaturing polyacrylamide gels in 0.5�Tris-boric acid-EDTA buffer at 100 V for 2 h at RT. The electrophoresed binding reactions were transferred to a nylon membrane (Hybond-N+; Amersham) by capillary transfer system overnight at RT. The biotin end-labeled DNA probe was detected using the conjugated streptavidin-horseradish peroxidase and chemiluminescent substrate. The membranes were exposed to -ray film for 2-5 min to obtain the perfect signal. Another consensus sequence, OCT-1 5'-TGTCGAATGCAAATCACTAGAA-3', was used as the control for the quantity and quality of nuclear extracts.
RNA preparation and reverse
transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from the ESCC cell lines with Trizol reagent according to the manufacturer's protocols and treated with the DNA-free kit to remove residual genomic DNA. Briefly, the isolated RNA (1 mg) was reverse transcribed to cDNA in a 20 ml reaction mixture containing 1 ml AMV reverse transcriptase, 1 ml random hexamer, 4 ml 5�AMV buffer, 1 ml RNase inhibitor (20 U/ml), 2 ml dNTP (10 mM) at 37 �C for 1 h. The cDNA mixture (2 ml) was used for the PCR amplification mixture (50 ml) containing 1.5 U Taq DNA polymerase in 10�PCR buffer, 1.5 mM MgCl2, 150 mM dNTP mixture, and 50 pmol of sense and antisense primers. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as an internal control. The following oligonucleotide primers were used for analysis of p65, p50 and IkBa amplification by PCR. For p65 (630 bp), forward primer 5'-ATAGAAGAGCAGCGTGGGGACT-3' and reverse primer 5'-GGATGACGTAAAGGGATAGGGC-3'; for p50 (449 bp), forward primer 5'-AAACCTTTCCTCTACTATCCTGA-3' and reverse primer 5'-GCACTCTCTTCTTTTGTTCCTGT-3'; for IkBa (316 bp), forward primer 5'-GAAGGAGCGGCTACTGGACG-3' and reverse primer 5'-AATTTCTGTGTGGCTGGTTGGTGA-3'; and for GAPDH (915 bp), forward primer 5'-AAGGTCGGAGTCAACGGATTTG-3' and reverse primer 5'-CTTGACAAAGTGGTCGTTGAGG-3'. PCR conditions: 4 min at 95 �C for initial denaturing, followed by 30 cycles of 95 �C for 1 min, annealing for 1 min at 55 �C for p65, 60 �C for p50 and 57 �C for IkBa, then 72 �C for 1 min and a final extension for 5 min at 72 �C. The amplified products were subjected to electrophoresis on 1% agarose gels.
RNAi
EC9706 (5�104 cells/well) and Eca109 (6�104 cells/well) cells were grown in six-well plates for 24 h in medium without antibiotics before siRNA transfection. The cells were transfected for 5 h with 8 ml of siRNA (10 mM) using 5 ml of transfection reagents. Subsequently, 0.8 ml of normal growth medium containing serum and antibodies was added to each well containing transfected cells without removing the transfection mixture. After the transfected cells were incubated for 72 h, they were collected to detect the expression of p65 proteins by Western blot analysis.
Cell proliferation assay
To test the effect of RNAi targeting at p65, EC9706 and Eca109 cells were transfected with p65 siRNA for 24 h in a 12-well plate. After the cells were incubated for 24 h in a 96-well plate at a concentration of 1�104 cells per well, the cells were treated with different concentrations of 5-FU for 24 or 48 h, respectively. The cells were then incubated with 20 ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (Sigma, St. Louis, USA) (at final concentration of 0.5 mg/ml) at 37 �C for 4 h. The medium was removed and the precipitated formazan was dissolved by adding 200 ml dimethylsulfoxide (Sigma). After the samples were placed on a shaker for 20-30 min, the absorbance at 570 nm was detected using a microplate reader.
Statistical analysis
Data were analyzed by statistical software spss version 13.0 (SPSS, Chicago, USA). Comparisons between the control and tested groups were performed using Student's t-test. Statistical results were expressed as means眘tandard deviations, except as otherwise stated. P<0.05 was considered statistically significant.
Results
Expression of NF-kB subunits in cytoplasmic extracts of
ESCC cell lines
The two ESCC cell lines had intense immunoreactivity to p50 and p65 in the cytoplasm by immunocytochemistry [Fig. 1(A)]. Western blot analysis of cytoplasmic extracts from the ESCC cell lines and the control cell line (HeLa229) investigated the expression levels of p50 and p65. As shown in Fig. 1, the two ESCC cell lines had the same NF-kB activity in the cytoplasm compared to the control cell line.
Expression and phosphorylation
status of IkBa
proteins
Because phosphorylation and degradation of IkBa are necessary for NF-kB activity, the expression level of IkBa protein and its phosphorylation status were detected by immunocytochemistry and Western blot to determine whether the high NF-kB activity in the two ESCC cell lines was caused by IkBa degradation. The results in the two ESCC cells were compared with the control cells. The two ESCC cell lines displayed different degree staining for IkBa and p-IkBa (Fig. 2). These results demonstrated that IkBa proteins in the ESCC cell lines were phosphorylated, suggesting that NF-kB activity was enhanced in the ESCC cells.
mRNA expression of p50, p65
and IkBa
in ESCC lines
The mRNA expressions of p50, p65 and IkBa in the two ESCC cell lines were analyzed by RT-PCR. As shown in Fig. 3, the mRNA expressions of p50, p65 and IkBa were detected in the ESCC cells. The results indicate that these genes might play roles in the maintenance of constitutive NF-kB activity in the two ESCC cell lines.
Increased NF-kB activity in ESCC lines
Western blot analysis of the nuclear extracts showed the same expression levels of p50 and p65 in the ESCC lines compared with control cells [Fig. 4(A)]. The NF-kB DNA-binding activity of nuclear protein extracts was analyzed by EMSA with a double-stranded biotin-labeled oligonucleotide probe containing a high-affinity kB sequence. The two ESCC cell lines showed a high level of NF-kB DNA-binding activity [Fig. 4(B)].
Expression of IKKb proteins in ESCC cells
IkBa phosphorylation is mediated by IKK proteins, in the upstream of the NF-kB signaling pathway, therefore we examined the expression of IKKb by immunocytochemical and Western blot analysis. IKKb proteins were expressed and localized in the cytoplasm in the two ESCC cell lines (Fig. 5), suggesting that the high levels of IKKb proteins might be another mechanism related to the increased NF-kB binding activity in ESCC cells.
Expression of p65 transfected
with p65 siRNA
To examine whether p65 expression could be effectively inhibited by siRNA after the cells had been transfected with p65 siRNA, the cells were harvested after transfection for 72 h and the expression of p65 protein in the cytoplasm was analyzed by Western blot. As shown in Fig. 6, the protein level of p65 decreased after transfection with p65 siRNA, whereas the protein level of MARK was not affected, suggesting that p65 siRNA can effectively inhibit p65 protein levels.
Effects of 5-FU at different
concentrations combined with p65 siRNA on cell proliferation
To determine whether a combined effect exists between p65 siRNA and 5-FU, we compared the growth rates of the two ESCC cell lines with or without transfection using siRNA at various of concentrations of 5-FU for 24 or 48 h. As shown in Fig. 7, after exposure to 5-FU for 24 or 48 h, both cells transfected with or without p65 siRNA were inhibited in a concentration-dependent manner. But at the same concentration of 5-FU, cells transfected with p65siRNA had a higher inhibitory effect than untransfected cells. These results suggest that p65 siRNA increases the sensitivity of 5-FU in the two ESCC cell lines.
Discussion
Aberrant activation of NF-kB has been shown in many cancers, such as pancreatic, cervical, head and neck cancers [10-12], and non-Hodgkin's lymphoma [13]. Some reports have revealed that NF-kB is an important regulator for several genes involved in the survival, transformation, differentiation, invasion and growth of tumor cells [14-16]. Constitutively activated NF-kB has been implicated in the carcinogenesis of those cancers. However, it is still unknown whether the NF-kB signaling pathway is related to carcinogenesis of the esophagus. This study demonstrated that the subunit of NF-kB in two ESCC cell lines, which is composed of p50 and p65, the most common heterodimer of NF-kB, could bind to DNA target sites. These results are consistent with the report published by Morceau et al. [17]. Furthermore, the overexpression of p50 and p65 proteins was detected in both the cytoplasm and the nucleus in the ESCC cell lines. The results of EMSA showed that NF-kB has high DNA binding activity in the two ESCC cell lines. The activated heterodimer of p50 and p65 in the cytoplasm was translocated to the nucleus and regulated the targeted genes. The NF-kB pathway was activated by a variety of stimuli, such as tumor necrosis factor-a, interleukin-1, ionizing radiation and cancer chemotherapeutic compounds. Without any possibility of inducing NF-kB activity in this study, the high NF-kB activity is a constitutive and intrinsic feature of the two ESCC cell lines. Due to the half-life of NF-kB being less than 30 min, the maintenance of its activity needs ongoing protein synthesis [18]. Upregulated transcription factor activity is frequently caused by gene overexpression [19]. In this study, the results of RT-PCR showed the overexpression of p50 and p65 mRNA in two ESCC cell lines. The expression of p50 and p65 was presumably a result of functional activation of NF-kB in the ESCC cell lines. Therefore, activated high levels of NF-kB in the ESCC cell lines mainly existed at translational, nuclear transporting and DNA-binding levels, consistent with the results of Wang and Cassidy [20].
It is well known that degradation and phosphorylation of IkB, a natural inhibitor of NF-kB in the cytoplasm, seems to be a critical reason to enhance NF-kB activation [21]. IkBa is an important element of the IkB family. Previous reports have identified that the cytoplasmic pool of IkB is degraded or phosphorylated by different stimuli factors, which are dissociated from the NF-kB-IkB complex, permitting the activated NF-kB to translocate to the nucleus and subsequently activate the expression of some important target genes [22-24]. In this study, we detected IkBa expression using immunocytochemistry, Western blot and RT-PCR. The IkBa and p-IkBa proteins had different expression levels in the cytoplasm of ESCC cell lines by immunocytochemistry and Western blot analysis. The increased expression of IkBa mRNA in the ESCC cell lines might be a feedback mechanism caused by the activated NF-kB, as the activation of NF-kB is known to lead to the upregulation of IkBa as a feedback in other cancers [25,26]. This suggests that upregulation of IkBa at the transcriptional level and degradation of IkBa play an indispensable role in the constitutively activated NF-kB signaling pathway.
The molecular basis of the activated NF-kB in many cancers is still not known. However, a well-known signaling pathway including the activity of IkB-kinase, which phosphorylates IkBa proteins, leads to its degradation [27,28]. IKKb, a representative of molecule upstream of the activation NF-kB signaling pathway, phosphorylates IkBa on serines 32 and 36 with approximately equal efficiency. By immunocytochemistry and Western blot analysis, the expression of IKKb protein was detected in the cytoplasm of the ESCC cell lines. Therefore, the overexpression of IKKb might be one of the important mechanisms involved in the constitutively activated NF-kB signaling pathway.
Activation of NF-kB seems to have many necessary functions, not only for tumor growth but also for invasion and metastasis, as well as for inducing the resistance of tumor cells to radiotherapy or chemotherapy [29-31]. Many studies using different inhibitors affecting the activated IKKb/NF-kB pathway have demonstrated that these methods have beneficial effects on tumor transformation or increase sensitivity to radiotherapy or chemotherapy [32-34]. RNAi has become a powerful strategy to knockdown and understand gene function. RNAi is a general mechanism for the sequence-specific gene silencing induced by double-stranded RNA [35]. RNAi is mediated by siRNA, a double-stranded RNA, which is approximately 21-23 nucleotides and is specific for the sequence of its target [36].
In this study, it was demonstrated that the expression of p65 was specifically inhibited by p65 siRNA. By combining siRNA targeting at p65 with different concentrations of 5-FU, the two ESCC cell lines became sensitive to 5-FU at lower concentrations than those transfected cells without siRNA, indicating that p65 siRNA with 5-FU treatment significantly inhibits cancer cell growth. The results of this study demonstrate that the activated NF-kB signaling pathway plays a crucial role in tumor cell growth, and suppression of p65 by siRNA increases the sensitivity of the ESCC cells to 5-FU. The constitutively activated NF-kB signaling pathway in the ESCC cell lines might also be an important mechanism responsible for the survival and proliferation of the ESCC cells. The amplification of p50, p65 and IkBa or the degradation of IkBa by IKKb might be the main cause to influence the activity of NF-kB. Using the p65 siRNA, and reducing NF-kB activity, the two ESCC cells show an enhanced sensitivity to anticancer agents. Further studies are underway to investigate the action of the NF-kB signaling pathway and to evaluate the potential use of the NF-kB signaling pathway as a specific target for therapeutic strategies in ESCC with high NF-kB activity.
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